Mutations in many synaptic genes are associated with autism spectrum disorders (ASDs), suggesting... more Mutations in many synaptic genes are associated with autism spectrum disorders (ASDs), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C-substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C-mutation on human neurons has not been investigated. Here, we generated human knock-in neurons with the NLGN3 R451C-mutation. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C-mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses. No significant cell death and endoplasmic reticulum stress were detected. Importantly, the augmentation of excitatory transm...
ABSTRACTHeterozygousNRXN1deletions constitute the most prevalent currently known single-gene muta... more ABSTRACTHeterozygousNRXN1deletions constitute the most prevalent currently known single-gene mutation predisposing to schizophrenia. Previous studies showed that engineered heterozygousNRXN1deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multi-center effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygousNRXN1deletions. We show that in neurons that were trans-differentiated from induced pluripotent stem cells derived from threeNRXN1-deletion patients, the same impairment in neurotransmitter release was observed as in engineeredNRXN1-deficient neurons. This impairment manifested as a decrease in spontaneous synaptic events and in evoked synaptic responses, and an alteration...
BackgroundThe OPRM1 A118G gene variant (N40D) encoding the µ-opioid receptor (MOR) has been assoc... more BackgroundThe OPRM1 A118G gene variant (N40D) encoding the µ-opioid receptor (MOR) has been associated with dependence on opiates and other abused drugs but its mechanism is unknown. With opioid abuse-related deaths rising at unprecedented rates, understanding these mechanisms may provide a path to therapy.MethodsSeven human induced pluripotent stem (iPS) cell lines from homozygous N40D subjects (4 with N40 and 3 with D40 variants) were generated and human induced neuronal cells (iNs) were derived from these iPS cell lines. Morphological, gene expression as well as synaptic physiology analyses were conducted in human iN cells carrying N40D MOR variants; Two pairs of isogenic pluripotent stem cells carrying N40D were generated using CRISPR/Cas9 genome-editing and iN cells derived from them were analyzed.ResultsInhibitory human neurons generated from subjects carrying N40D MOR gene variants show mature properties in morphological and functional analyses. Gene expression revealed that ...
Glucagon-like Peptide 1 (GLP-1)-expressing neurons in the hindbrain send robust projections to th... more Glucagon-like Peptide 1 (GLP-1)-expressing neurons in the hindbrain send robust projections to the paraventricular nucleus of the hypothalamus (PVN), which is involved in the regulation of food intake. Here, we describe that stimulation of GLP-1 afferent fibers within the PVN is sufficient to suppress food intake independent of glutamate release. We also show that GLP-1 receptor (GLP-1R) activation augments excitatory synaptic strength in PVN corticotropin-releasing hormone (CRH) neurons, with GLP-1R activation promoting a protein kinase A (PKA)-dependent signaling cascade leading to phosphorylation of serine S845 on GluA1 AMPA receptors and their trafficking to the plasma membrane. Finally, we show that postnatal depletion of GLP-1R in the PVN increases food intake and causes obesity. This study provides a comprehensive multi-level (circuit, synaptic, and molecular) explanation of how food intake behavior and body weight are regulated by endogenous central GLP-1.
Rapid advances in genetics are linking mutations on genes to diseases at an exponential rate, yet... more Rapid advances in genetics are linking mutations on genes to diseases at an exponential rate, yet characterizing the gene mutation-cell behavior relationships essential for precision medicine remains a daunting task. More than 350 mutations on small GTPase BRaf are associated with various tumors, and ∼40 mutations are associated with the neurodevelopmental disorder cardio-facio-cutaneous syndrome (CFC). We developed a fast cost-effective lentivirus-based rapid gene replacement method to interrogate the physiopathology of BRaf and ∼50 disease-linked BRaf mutants, including all CFC-linked mutants. Analysis of simultaneous multiple patch-clamp recordings from 6068 pairs of rat neurons with validation in additional mouse and human neurons and multiple learning tests from 1486 rats identified BRaf as the key missing signaling effector in the common synaptic NMDA-R-CaMKII-SynGap-Ras-BRaf-MEK-ERK transduction cascade. Moreover, the analysis creates the original big data unveiling three gen...
Genetic variation in nicotinic receptor alpha 5 (CHRNA5) has been associated with increased risk ... more Genetic variation in nicotinic receptor alpha 5 (CHRNA5) has been associated with increased risk of addiction-associated phenotypes in humans yet little is known the underlying neural basis. Induced pluripotent stem cells (iPSCs) were derived from donors homozygous for either the major (D398) or the minor (N398) allele of the nonsynonymous single nucleotide polymorphism (SNP), rs16969968, in CHRNA5. To understand the impact of these nicotinic receptor variants in humans, we differentiated these iPSCs to dopamine (DA) or glutamatergic neurons and then tested their functional properties and response to nicotine. Results show that N398 variant human DA neurons differentially express genes associated with ligand receptor interaction and synaptic function. While both variants exhibited physiological properties consistent with mature neuronal function, the N398 neuronal population responded more actively with an increased excitatory postsynaptic current response upon the application of ni...
Two families of Ca(2+)-binding proteins have been proposed as Ca(2+) sensors for spontaneous rele... more Two families of Ca(2+)-binding proteins have been proposed as Ca(2+) sensors for spontaneous release: synaptotagmins and Doc2s, with the intriguing possibility that Doc2s may represent high-affinity Ca(2+) sensors that are activated by deletion of synaptotagmins, thereby accounting for the increased spontaneous release in synaptotagmin-deficient synapses. Here, we use an shRNA-dependent quadruple knockdown of all four Ca(2+)-binding proteins of the Doc2 family to confirm that Doc2-deficient synapses exhibit a marked decrease in the frequency of spontaneous release events. Knockdown of Doc2s in synaptotagmin-1-deficient synapses, however, failed to reduce either the increased spontaneous release or the decreased evoked release of these synapses, suggesting that Doc2s do not constitute Ca(2+) sensors for asynchronous release. Moreover, rescue experiments revealed that the decrease in spontaneous release induced by the Doc2 knockdown in wild-type synapses is fully reversed by mutant Do...
Synaptotagmin-1, the Ca2+sensor for fast neurotransmitter release, was proposed to function by Ca... more Synaptotagmin-1, the Ca2+sensor for fast neurotransmitter release, was proposed to function by Ca2+-dependent phospholipid binding and/or by Ca2+-dependent solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex binding. Extensivein vivodata support the first hypothesis, but testing the second hypothesis has been difficult because no synaptotagmin-1 mutation is known that selectively interferes with SNARE complex binding. Using knock-in mice that carry aspartate-to-asparagine substitutions in a Ca2+-binding site of synaptotagmin-1 (the D232N or D238N substitutions), we now show that the D232N mutation dramatically increases Ca2+-dependent SNARE complex binding by native synaptotagmin-1, but leaves phospholipid binding unchanged. In contrast, the adjacent D238N mutation does not significantly affect SNARE complex binding, but decreases phospholipid binding. Electrophysiological recordings revealed that the D232N mutation increased Ca2+-triggered release, ...
Calmodulin regulates multifarious cellular processes via a panoply of target interactions. Howeve... more Calmodulin regulates multifarious cellular processes via a panoply of target interactions. However, the central role, multiple isoforms, and complex target interactions of calmodulin make it difficult to examine its precise functions. Here, we analyzed calmodulin function in neurons using lentivirally delivered short-hairpin RNAs that suppressed expression of all calmodulin isoforms by ∼70%. Calmodulin knockdown did not significantly alter neuronal survival or synapse formation but depressed spontaneous neuronal network activity. Strikingly, calmodulin knockdown decreased the presynaptic release probability almost twofold, without altering the presynaptic readily-releasable vesicle pool or postsynaptic neurotransmitter reception. In calmodulin knockdown neurons, presynaptic release was restored to wild-type levels by expression of constitutively active calmodulin-dependent kinase-IIα (CaMKIIα); in contrast, in control neurons, expression of constitutively active CaMKIIα had no effec...
Although the calcium/calmodulin-activated phosphatase calcineurin may dephosphorylate many endocy... more Although the calcium/calmodulin-activated phosphatase calcineurin may dephosphorylate many endocytic proteins, it is not considered a key molecule in mediating the major forms of endocytosis at synapses—slow, clathrin-dependent and the rapid, clathrin-independent endocytosis. Here we studied the role of calcineurin in endocytosis by reducing calcium influx, inhibiting calmodulin with pharmacological blockers and knockdown of calmodulin, and by inhibiting calcineurin with pharmacological blockers and knock-out of calcineurin. These manipulations significantly inhibited both rapid and slow endocytosis at the large calyx-type synapse in 7- to 10-d-old rats and mice, and slow, clathrin-dependent endocytosis at the conventional cultured hippocampal synapse of rats and mice. These results suggest that calcium influx during nerve firing activates calcium/calmodulin-dependent calcineurin, which controls the speed of both rapid and slow endocytosis at synapses by dephosphorylating endocytic ...
Synaptotagmins comprise a large protein family, of which synaptotagmin 1 (Syt1) is a Ca2+sensor f... more Synaptotagmins comprise a large protein family, of which synaptotagmin 1 (Syt1) is a Ca2+sensor for fast exocytosis, and its close relative, synaptotagmin 2 (Syt2), is assumed to serve similar functions. Chromaffin cells express Syt1 but not Syt2. We compared secretion from chromaffin cells from Syt1 null mice overexpressing either Syt isoform. High time-resolution capacitance measurement showed that Syt1 null cells lack the exocytotic phase corresponding to the readily-releasable pool (RRP) of vesicles. Comparison with the amperometric signal confirmed that the missing phase of exocytosis consists of catecholamine-containing vesicles. Overexpression of Syt1 rescued the RRP and increased its size above wild-type values, whereas the size of the slowly releasable pool decreased, indicating that the availability of Syt1 regulates the relative size of the two releasable pools. The RRP was also rescued by Syt2 overexpression, but the kinetics of fusion was slightly slower than in cells e...
Proceedings of the National Academy of Sciences, 2013
Significance Synaptic vesicle fusion is catalyzed by multiprotein complexes that bring two lipid ... more Significance Synaptic vesicle fusion is catalyzed by multiprotein complexes that bring two lipid bilayers into close opposition. Several assembly mechanisms have been proposed for the synaptic vesicle fusion machinery, but exactly how these proteins interact in vivo remains unclear. We developed two-color fluorescence nanoscopy to directly visualize molecular interactions in situ and discovered that syntaxin-1, SNAP-25, and Munc18-1 (mammalian uncoordinated-18), three essential components for neurotransmission, closely colocalize on the plasma membrane, suggesting possible pathways for SNARE-mediated membrane fusion. Our superresolution method provides a framework for delineating the molecular underpinnings of the synaptic vesicle fusion machinery.
Mutations in many synaptic genes are associated with autism spectrum disorders (ASDs), suggesting... more Mutations in many synaptic genes are associated with autism spectrum disorders (ASDs), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C-substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C-mutation on human neurons has not been investigated. Here, we generated human knock-in neurons with the NLGN3 R451C-mutation. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C-mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses. No significant cell death and endoplasmic reticulum stress were detected. Importantly, the augmentation of excitatory transm...
ABSTRACTHeterozygousNRXN1deletions constitute the most prevalent currently known single-gene muta... more ABSTRACTHeterozygousNRXN1deletions constitute the most prevalent currently known single-gene mutation predisposing to schizophrenia. Previous studies showed that engineered heterozygousNRXN1deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multi-center effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygousNRXN1deletions. We show that in neurons that were trans-differentiated from induced pluripotent stem cells derived from threeNRXN1-deletion patients, the same impairment in neurotransmitter release was observed as in engineeredNRXN1-deficient neurons. This impairment manifested as a decrease in spontaneous synaptic events and in evoked synaptic responses, and an alteration...
BackgroundThe OPRM1 A118G gene variant (N40D) encoding the µ-opioid receptor (MOR) has been assoc... more BackgroundThe OPRM1 A118G gene variant (N40D) encoding the µ-opioid receptor (MOR) has been associated with dependence on opiates and other abused drugs but its mechanism is unknown. With opioid abuse-related deaths rising at unprecedented rates, understanding these mechanisms may provide a path to therapy.MethodsSeven human induced pluripotent stem (iPS) cell lines from homozygous N40D subjects (4 with N40 and 3 with D40 variants) were generated and human induced neuronal cells (iNs) were derived from these iPS cell lines. Morphological, gene expression as well as synaptic physiology analyses were conducted in human iN cells carrying N40D MOR variants; Two pairs of isogenic pluripotent stem cells carrying N40D were generated using CRISPR/Cas9 genome-editing and iN cells derived from them were analyzed.ResultsInhibitory human neurons generated from subjects carrying N40D MOR gene variants show mature properties in morphological and functional analyses. Gene expression revealed that ...
Glucagon-like Peptide 1 (GLP-1)-expressing neurons in the hindbrain send robust projections to th... more Glucagon-like Peptide 1 (GLP-1)-expressing neurons in the hindbrain send robust projections to the paraventricular nucleus of the hypothalamus (PVN), which is involved in the regulation of food intake. Here, we describe that stimulation of GLP-1 afferent fibers within the PVN is sufficient to suppress food intake independent of glutamate release. We also show that GLP-1 receptor (GLP-1R) activation augments excitatory synaptic strength in PVN corticotropin-releasing hormone (CRH) neurons, with GLP-1R activation promoting a protein kinase A (PKA)-dependent signaling cascade leading to phosphorylation of serine S845 on GluA1 AMPA receptors and their trafficking to the plasma membrane. Finally, we show that postnatal depletion of GLP-1R in the PVN increases food intake and causes obesity. This study provides a comprehensive multi-level (circuit, synaptic, and molecular) explanation of how food intake behavior and body weight are regulated by endogenous central GLP-1.
Rapid advances in genetics are linking mutations on genes to diseases at an exponential rate, yet... more Rapid advances in genetics are linking mutations on genes to diseases at an exponential rate, yet characterizing the gene mutation-cell behavior relationships essential for precision medicine remains a daunting task. More than 350 mutations on small GTPase BRaf are associated with various tumors, and ∼40 mutations are associated with the neurodevelopmental disorder cardio-facio-cutaneous syndrome (CFC). We developed a fast cost-effective lentivirus-based rapid gene replacement method to interrogate the physiopathology of BRaf and ∼50 disease-linked BRaf mutants, including all CFC-linked mutants. Analysis of simultaneous multiple patch-clamp recordings from 6068 pairs of rat neurons with validation in additional mouse and human neurons and multiple learning tests from 1486 rats identified BRaf as the key missing signaling effector in the common synaptic NMDA-R-CaMKII-SynGap-Ras-BRaf-MEK-ERK transduction cascade. Moreover, the analysis creates the original big data unveiling three gen...
Genetic variation in nicotinic receptor alpha 5 (CHRNA5) has been associated with increased risk ... more Genetic variation in nicotinic receptor alpha 5 (CHRNA5) has been associated with increased risk of addiction-associated phenotypes in humans yet little is known the underlying neural basis. Induced pluripotent stem cells (iPSCs) were derived from donors homozygous for either the major (D398) or the minor (N398) allele of the nonsynonymous single nucleotide polymorphism (SNP), rs16969968, in CHRNA5. To understand the impact of these nicotinic receptor variants in humans, we differentiated these iPSCs to dopamine (DA) or glutamatergic neurons and then tested their functional properties and response to nicotine. Results show that N398 variant human DA neurons differentially express genes associated with ligand receptor interaction and synaptic function. While both variants exhibited physiological properties consistent with mature neuronal function, the N398 neuronal population responded more actively with an increased excitatory postsynaptic current response upon the application of ni...
Two families of Ca(2+)-binding proteins have been proposed as Ca(2+) sensors for spontaneous rele... more Two families of Ca(2+)-binding proteins have been proposed as Ca(2+) sensors for spontaneous release: synaptotagmins and Doc2s, with the intriguing possibility that Doc2s may represent high-affinity Ca(2+) sensors that are activated by deletion of synaptotagmins, thereby accounting for the increased spontaneous release in synaptotagmin-deficient synapses. Here, we use an shRNA-dependent quadruple knockdown of all four Ca(2+)-binding proteins of the Doc2 family to confirm that Doc2-deficient synapses exhibit a marked decrease in the frequency of spontaneous release events. Knockdown of Doc2s in synaptotagmin-1-deficient synapses, however, failed to reduce either the increased spontaneous release or the decreased evoked release of these synapses, suggesting that Doc2s do not constitute Ca(2+) sensors for asynchronous release. Moreover, rescue experiments revealed that the decrease in spontaneous release induced by the Doc2 knockdown in wild-type synapses is fully reversed by mutant Do...
Synaptotagmin-1, the Ca2+sensor for fast neurotransmitter release, was proposed to function by Ca... more Synaptotagmin-1, the Ca2+sensor for fast neurotransmitter release, was proposed to function by Ca2+-dependent phospholipid binding and/or by Ca2+-dependent solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex binding. Extensivein vivodata support the first hypothesis, but testing the second hypothesis has been difficult because no synaptotagmin-1 mutation is known that selectively interferes with SNARE complex binding. Using knock-in mice that carry aspartate-to-asparagine substitutions in a Ca2+-binding site of synaptotagmin-1 (the D232N or D238N substitutions), we now show that the D232N mutation dramatically increases Ca2+-dependent SNARE complex binding by native synaptotagmin-1, but leaves phospholipid binding unchanged. In contrast, the adjacent D238N mutation does not significantly affect SNARE complex binding, but decreases phospholipid binding. Electrophysiological recordings revealed that the D232N mutation increased Ca2+-triggered release, ...
Calmodulin regulates multifarious cellular processes via a panoply of target interactions. Howeve... more Calmodulin regulates multifarious cellular processes via a panoply of target interactions. However, the central role, multiple isoforms, and complex target interactions of calmodulin make it difficult to examine its precise functions. Here, we analyzed calmodulin function in neurons using lentivirally delivered short-hairpin RNAs that suppressed expression of all calmodulin isoforms by ∼70%. Calmodulin knockdown did not significantly alter neuronal survival or synapse formation but depressed spontaneous neuronal network activity. Strikingly, calmodulin knockdown decreased the presynaptic release probability almost twofold, without altering the presynaptic readily-releasable vesicle pool or postsynaptic neurotransmitter reception. In calmodulin knockdown neurons, presynaptic release was restored to wild-type levels by expression of constitutively active calmodulin-dependent kinase-IIα (CaMKIIα); in contrast, in control neurons, expression of constitutively active CaMKIIα had no effec...
Although the calcium/calmodulin-activated phosphatase calcineurin may dephosphorylate many endocy... more Although the calcium/calmodulin-activated phosphatase calcineurin may dephosphorylate many endocytic proteins, it is not considered a key molecule in mediating the major forms of endocytosis at synapses—slow, clathrin-dependent and the rapid, clathrin-independent endocytosis. Here we studied the role of calcineurin in endocytosis by reducing calcium influx, inhibiting calmodulin with pharmacological blockers and knockdown of calmodulin, and by inhibiting calcineurin with pharmacological blockers and knock-out of calcineurin. These manipulations significantly inhibited both rapid and slow endocytosis at the large calyx-type synapse in 7- to 10-d-old rats and mice, and slow, clathrin-dependent endocytosis at the conventional cultured hippocampal synapse of rats and mice. These results suggest that calcium influx during nerve firing activates calcium/calmodulin-dependent calcineurin, which controls the speed of both rapid and slow endocytosis at synapses by dephosphorylating endocytic ...
Synaptotagmins comprise a large protein family, of which synaptotagmin 1 (Syt1) is a Ca2+sensor f... more Synaptotagmins comprise a large protein family, of which synaptotagmin 1 (Syt1) is a Ca2+sensor for fast exocytosis, and its close relative, synaptotagmin 2 (Syt2), is assumed to serve similar functions. Chromaffin cells express Syt1 but not Syt2. We compared secretion from chromaffin cells from Syt1 null mice overexpressing either Syt isoform. High time-resolution capacitance measurement showed that Syt1 null cells lack the exocytotic phase corresponding to the readily-releasable pool (RRP) of vesicles. Comparison with the amperometric signal confirmed that the missing phase of exocytosis consists of catecholamine-containing vesicles. Overexpression of Syt1 rescued the RRP and increased its size above wild-type values, whereas the size of the slowly releasable pool decreased, indicating that the availability of Syt1 regulates the relative size of the two releasable pools. The RRP was also rescued by Syt2 overexpression, but the kinetics of fusion was slightly slower than in cells e...
Proceedings of the National Academy of Sciences, 2013
Significance Synaptic vesicle fusion is catalyzed by multiprotein complexes that bring two lipid ... more Significance Synaptic vesicle fusion is catalyzed by multiprotein complexes that bring two lipid bilayers into close opposition. Several assembly mechanisms have been proposed for the synaptic vesicle fusion machinery, but exactly how these proteins interact in vivo remains unclear. We developed two-color fluorescence nanoscopy to directly visualize molecular interactions in situ and discovered that syntaxin-1, SNAP-25, and Munc18-1 (mammalian uncoordinated-18), three essential components for neurotransmission, closely colocalize on the plasma membrane, suggesting possible pathways for SNARE-mediated membrane fusion. Our superresolution method provides a framework for delineating the molecular underpinnings of the synaptic vesicle fusion machinery.
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